Multi-epitope vaccine antigen based on BCG and application thereof

Abstract

The invention provides a multi-epitope vaccine antigen based on BCG, which comprises a recombinant epitope polypeptide sequence formed by sequentially connecting a CD8 + T cell epitope (CTL) polypeptide sequence, a CD4 + T cell epitope (HTL) polypeptide sequence, a B cell epitope (LBEs) polypeptide sequence and a B cell conformational epitope (CBEs) polypeptide sequence in series, the multi-epitope vaccine antigen provided by the invention can have good recognition and binding effects with an immune related receptor, is an effective immune antigen target, has a relatively strong immune effect, has a remarkable effect on humoral immune level and cellular immune level induced in a mouse body, and is higher than BCG immune level.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a BCG-based multi-epitope vaccine antigen and application thereof. Background Art

[0002] Against the backdrop of the global novel coronavirus pandemic, more than 10 million new cases of tuberculosis are reported worldwide each year, and the incidence of tuberculosis increased for the first time in 20 years in 2021. Despite efforts to combat the disease, the global burden of tuberculosis is expected to increase in the future.

[0003] Bacillus Calmette-Guérin (BCG), derived from Mycobacterium bovis, is currently the only approved TB vaccine. However, as it is a live attenuated vaccine, its effectiveness in adults remains a controversial topic. As of September 2022, there are 17 candidate vaccines in various stages of clinical trials, and progress in developing new vaccines is below the target set by the WHO End TB Strategy. To effectively address the challenges posed by TB, there is an urgent need to accelerate the development and deployment of innovative prevention and control strategies and new vaccines.

[0004] Traditionally, vaccines are produced using live or attenuated microorganisms or their parts. However, the presence of immunologically redundant biological components or biological impurities in such vaccines may cause serious problems. Peptide vaccines offer an attractive alternative strategy that relies on the use of short peptide fragments to induce highly targeted immune responses while avoiding adverse immune responses. In addition, Mycobacterium tuberculosis cellular immunity has been shown to be more effective in protecting the host from tuberculosis infection, and epitope-based vaccines are specific and accurate. Therefore, epitopes that stimulate CD4+ and CD8+ T cells may be ideal considerations in the design of peptide-based vaccines.

[0005] The secretion system of Mtb, particularly the type VII secretion system (T7SS), is essential for the secretion of Mtb effector proteins and the establishment of host-pathogen interactions, making it a potential therapeutic target. These secreted proteins can modulate cellular functions throughout the host. Mtb encodes five T7SS systems, designated ESX-1 to ESX-5, which are essential for growth and pathogenesis; among them, ESX-1, -3, and -5 are essential for mycobacterial virulence. ESAT-6 (also known as esxA), located within the RD1 locus, is absent in the M. bovis BCG strain used for vaccination and is considered a major factor contributing to the reduced efficacy of BCG. As the most potent T cell antigen secreted by Mtb, ESAT-6 plays multiple roles in regulating the host immune response to infection, including the suppression of host T cell production of IFN-γ, IL-17, and TNF-α. In addition, ESAT-6 may help regulate the assembly and function of the T7SS and is considered a potential vaccine candidate. PE35, a member of the PE_PPE protein family, is another important antigen encoded by a gene within the RD1 region and is required for ESAT-617 expression. PE4 is a protein secreted by ESX-3 that is involved in iron acquisition and overcoming host-induced iron limitation. PE4 has been shown to show increased expression during Mtb infection and is considered an immunodominant antigen that can elicit a strong humoral response in patients. The substrates of ESX-5, PPE-MPTR and PE_PGRS, which contain more than 80 proteins, are affected by the PPE3820 mutation, resulting in altered secretion and reduced proinflammatory responses in infected macrophages. These findings suggest that proteins secreted by the T7SS in various Mtb strains can serve as valuable candidates for epitope vaccine development.

[0006] However, faced with numerous protein families and regions, there is currently no report on how to select and apply them to obtain a safe and effective BCG vaccine. Summary of the invention

[0007] In order to overcome the above technical problems, the present invention provides a multi-gene engineering vaccine with good binding performance with tuberculosis infected serum, high safety and good immunogenicity and its application.

[0008] Specifically, the present invention provides a BCG-based multi-epitope vaccine antigen, which comprises a recombinant epitope polypeptide sequence composed of a CD8+T cell epitope (CTL) polypeptide, a CD4+T cell epitope (HTL) polypeptide, a B cell epitope (LBEs) polypeptide, and a B cell conformational epitope (CBEs) polypeptide sequence connected in series.

[0009] Wherein, the CD8+T cell epitope (CTL) polypeptide is composed of the following amino acid sequences connected in series:

[0010] SEQ ID No. 1SEAYQGVQQK;

[0011] SEQ ID No. 2EAYQGVQQKW;

[0012] SEQ ID No. 3ASTEGNVTGM;

[0013] SEQ ID No. 4GTQVSDNALH;

[0014] SEQ ID No. 5NASAQDQLHR;

[0015] SEQ ID No. 6GANPHVVQAR;

[0016] SEQ ID No. 7VQARPSVVPR;

[0017] The CD4+T cell epitope (HTL) polypeptide is composed of the following amino acid sequences connected in series:

[0018] SEQ ID No. 8QQWNFAGIEAAASA;

[0019] SEQ ID No. 9TEQQWNFAGIEAAAS;

[0020] SEQ ID No.10AGNAYAHAEAANAAR;

[0021] SEQ ID No.11GNAYAHAEAANAARV;

[0022] The B cell epitope (LBEs) polypeptide is composed of the following amino acid sequences connected in series:

[0023] SEQ ID No.12GGSGSEAYQGVQQKWD;

[0024] SEQ ID No.13EKMSHDPIAADIGTQV;

[0025] SEQ ID No.14PEINSARIYAGAGSGP;

[0026] SEQ ID No.15GAGMPAGMMGRGGANP;

[0027] SEQ ID No.16PDSYPWVPSINPGLNF;

[0028] SEQ ID No.17VPSINPGLNFYFGQPQ;

[0029] The B cell conformational epitope (CBEs) polypeptide is composed of the following amino acid sequences connected in series:

[0030] SEQ ID No. 18NIRQAGVQYS;

[0031] SEQ ID No. 19AAWGGSGSEAYQ;

[0032] SEQ ID No. 20VQYSRADEEQ;

[0033] SEQ ID No. 21MAEMKTDAAT;

[0034] SEQ ID No.22STAGSLQGQWRGAAGTAAQAAVVRFQE;

[0035] SEQ ID No. 23AAWGGSGSEAYQGVQQ.

[0036] In an optional embodiment, the recombinant epitope polypeptide sequence further comprises a connecting peptide sequence.

[0037] In an optional embodiment, the connecting peptide is used to connect in series the polypeptide sequence in the CD8+ T cell epitope (CTL), and optionally, the amino acid sequence of the connecting peptide is AAY (SEQ ID No. 24).

[0038] In an optional embodiment, the connecting peptide is used to connect in series the polypeptide sequence in the CD4+ T cell epitope (HTL), and optionally, the amino acid sequence of the connecting peptide is GPGPG (SEQ ID No. 25).

[0039] In an optional embodiment, the connecting peptide is used to connect polypeptide sequences in tandem B cell epitopes (LBEs), and optionally, the amino acid sequence of the connecting peptide is KK (SEQ ID No. 26).

[0040] In an optional embodiment, the connecting peptide is used to connect polypeptide sequences in tandem in B cell conformational epitopes (CBEs), and optionally, the amino acid sequence of the connecting peptide is KK (SEQ ID No. 26).

[0041] In an optional embodiment, the CD8+ T cell epitope (CTL) polypeptide and the CD4+ T cell epitope (HTL) polypeptide sequence are connected by a connecting peptide, and the amino acid sequence of the connecting peptide is HEYGAEACERAG (SEQ ID No. 27).

[0042] In an optional embodiment, the CD4+T cell epitope (HTL) polypeptide sequence and the B cell epitope (LBEs) polypeptide, and the B cell epitope (LBEs) polypeptide and the B cell conformational epitope (CBEs) are connected by a connecting peptide, and the amino acid sequence of the connecting peptide is KK (SEQ ID No.26).

[0043] In an optional embodiment, the recombinant epitope polypeptide further comprises an immunomodulatory sequence. Preferably, the immunomodulatory sequence is a human β-defensin-3 adjuvant sequence, and its sequence is shown in SEQ ID No.28.

[0044] Furthermore, the immunomodulatory sequence is connected to the N-terminus of the recombinant epitope polypeptide using a connecting peptide, and the connecting peptide sequence is EAAAK (SEQ ID No. 29).

[0045] In an optional embodiment, the recombinant epitope polypeptide further comprises an adjuvant antibody response sequence, and the adjuvant antibody response sequence is a PADRE sequence, and its sequence is shown in SEQ ID No.30.

[0046] Furthermore, the adjuvant antibody response sequence is connected to the immunomodulatory sequence with a connecting peptide, and the connecting peptide is EAAAK (SEQ ID No. 29).

[0047] In an optional embodiment, the recombinant epitope polypeptide further comprises a his-tag purification tag, and the tag sequence is HHHHHH (SEQ ID No. 31).

[0048] In an optional embodiment, the recombinant epitope polypeptide sequence in the BCG multi-epitope vaccine is shown as SEQ ID No.32.

[0049] The present invention further provides the nucleotide sequence of the recombinant epitope polypeptide in the BCG multi-epitope vaccine, as shown in SEQ ID No.33.

[0050] The present invention provides the use of the above-mentioned recombinant epitope polypeptide in the preparation of a BCG prevention or treatment drug. Preferably, the BCG prevention or treatment drug is a multi-epitope vaccine.

[0051] The present invention also provides a BCG multi-epitope vaccine, which comprises the above-mentioned recombinant epitope polypeptide and a pharmaceutically acceptable carrier or reagent.

[0052] Furthermore, the present invention also provides a method for preparing the above-mentioned recombinant epitope polypeptide, the steps of which include cloning the above-mentioned epitope polypeptide sequence into the mycobacterium plasmid pMV361, transforming it into the BCG strain by electroporation, culturing and amplifying it for 2-3 weeks, inducing it, collecting the culture and purifying it.

[0053] Compared with the prior art, the present invention has the following beneficial effects:

[0054] 1. The recombinant epitope polypeptide of the present invention can dock with TLR4 at multiple points and has a stable and close interaction, indicating that the epitope peptide can have good recognition and binding effects with immune-related receptors and is an effective immune antigen target. It also has a strong ability to bind to TLR-4 (Toll-like receptors) receptors and can effectively activate immune responses.

[0055] 2. The recombinant epitope polypeptide of the present invention can detect tuberculosis in the serum of tuberculosis patients with good specificity and strong immune effect. The humoral immunity level and cellular immunity level induced in mice have significant effects and are higher than the BCG immunity level.

[0056] 3. The recombinant epitope polypeptide of the present invention has good hydrophilicity and immunogenicity, and is non-sensitizing, stable, non-toxic, and has good stability and safety.

[0057] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 This is a comparison of the solubility of tMEV protein in Example 1;

[0059] Figure 2 The visualized protein interaction diagram in Example 1;

[0060] Figure 3 This is a simulation diagram of the interaction between tMEV and TLR-4 receptor in Example 1;

[0061] Figure 4 The Western blot test results of tMEV in Example 2 are shown in Figure A, where A is the test result of serum of tuberculosis patients, and B is the test result of serum of normal people;

[0062] Figure 5The IgG1 and IgG2a antibody titers of the mice serum in the PBS group, tMEV group, BCG group and rBCG-tMEV group were analyzed in Example 3. A. The ratio of IgG1 to IgG2a in the mice serum in the PBS group, tMEV group, BCG group and rBCG-tMEV group, where the blue dotted line is the ratio of IgG1 to IgG2a in the PBS group of this study; B. The logarithmic value of the IgG1 antibody titer of the mice serum in the PBS group, tMEV group, BCG group and rBCG-tMEV group; C. The logarithmic value of the IgG2a antibody titer of the mice serum in the PBS group, tMEV group, BCG group and rBCG-tMEV group;

[0063] Figure 6 This is the T cell activation analysis of the PBS group, tMEV group, BCG group, and rBCG-tMEV group in Example 3. A. The left side shows the statistical data of the percentage of CD4+T cells to the percentage of CD3+T cells; the right side shows the percentage of CD4+T cells to CD3+T cells; B. The left side shows the statistical data of the percentage of CD8+T cells to the ratio of CD3+T cells; the right side shows the percentage of CD8+T cells to CD3+T cells.

[0064] Figure 7 The bacterial counts in the spleen and lungs of mice were measured 8 weeks after the challenge experiment in Example 3. The left picture shows the lung load of mice infected with Mycobacterium tuberculosis 8 weeks after inoculation; the right picture shows the spleen load of mice infected with Mycobacterium tuberculosis 8 weeks after inoculation.

[0065] Figure 8 The results of the secretion detection of IFN-γ cytokines and IL-2 cytokines in the PBS group, tMEV group, BCG group and rBCG-tMEV group in Example 3 are shown in Figure 3; A. The left side is a statistical curve of the secretion of IFN-γ cytokines in mice two weeks after the last vaccination; the right side is an ELISPOT spot diagram, corresponding to the results of three replicates of one mouse. B. The left side is a statistical graph of the secretion of IL-2 cytokines in mice 2 weeks after the last vaccination; the right side is an ELISPOT spot diagram, corresponding to the results of three replicates of one mouse;

[0066] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. DETAILED DESCRIPTION

[0067] If no specific conditions are specified in the examples of the present invention, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0068] Example 1 Preparation method of recombinant epitope antigen tMEV

[0069] 1.1 Construction of recombinant plasmid pMV361-tMEV

[0070] The tMEV gene was amplified from the constructed pET41a-tMEV plasmid, and the purified 1416 bp tMEV fragment was subcloned into the Escherichia coli-Mycobacterium shuttle plasmid pMV361 using EcoRI and HindIII restriction enzymes.

[0071] 1.2 Construction of rBCG-tMEV plasmid

[0072] The recombinant plasmid pMV361-tMEV was transformed into BCG by electroporation (Electroporator, Eppendorf). The transformed bacteria were placed on Middlebrook 7H10 agar supplemented with 10% oleic acid albumin-dextrose-catalase (OADC) and 0.5% Tween 80, and incubated at 37°C for 4 weeks in a selective medium containing 30ug / ml kanamycin. After selection, the transformed bacteria were further cultured and amplified in Middlebrook 7H9 liquid medium for 2-3 weeks.

[0073] 1.3 Expression of rBCG-tMEV

[0074] BCG was cultured in 7H9 liquid medium containing 10% oleic acid albumin-glucose-catalase (OADC), 0.05% Tween 80 and 30 mg / ml kanamycin for about 2 weeks. After the A600 value reached 0.6-0.8, it was incubated at 37°C for 2 weeks. Then the induction culture was continued for 7 days. After culture, centrifuged at 12,000 rpm for 15 minutes at 4°C. Washed twice with phosphate buffered saline (PBS), lysis buffer was added, and treated with protease inhibitors (PMSF). After that, ultrasonic disruption was performed in an ice bath for 30 minutes, and then centrifuged at 12,000 rpm for 1 hour. The supernatant was collected, the protein was separated using 10% SDS-PAGE gel, and the his-tagged recombinant protein was purified by HisTrap HP (Cytiva).

[0075] Example 2 Analysis of the properties of the recombinant epitope antigen tMEV

[0076] 1.1 Analysis of tMEV solubility, safety, stability and antigenicity

[0077] We predicted protein solubility using Protein-sol (https: / / protein-sol.manchester.ac.uk), a tool that uses the solubility data of E. coli proteins in cell-free expression systems to predict the protein solubility of the sequence. The results showed that tMEV protein has a higher solubility than E. coli protein (results as shown in Figure 1 In order to improve the accuracy of solubility prediction, SOLpro (http: / / scratch.proteomics.ics.uci.edu / ) was used for further prediction. SOLpro software predicted that its MEV solubility was 0.759, which further supported its good solubility (the results are shown in Tables 1-3). At the same time, the prediction results showed that it had good hydrophilicity and immunogenicity, and was non-sensitizing, stable, and non-toxic.

[0078] Table 1 Analysis of solubility, safety, stability and antigenicity of B cell conformational epitope peptides

[0079]

[0080]

[0081] Table 2 Analysis of solubility, safety and antigenicity of B cell epitope peptides

[0082]

[0083] Table 3 Analysis of solubility, safety, stability and antigenicity of CD8+ T cell epitope and CD4+ T cell epitope peptides

[0084]

[0085] 1.2 Molecular docking performance analysis

[0086] TLR4 is a transmembrane receptor present in the cytoplasm and vesicle membranes, which is used to recognize a variety of molecules, including BCG, and is the molecular docking site of tMEV. The more docking sites there are on the tMEV binding, the stronger its ability to interact with TLR4 is, thereby triggering innate immunity and making the immune response move towards Th1 immunity, which is the key immune direction against mycobacterial infection.

[0087] The structures of tMEV protein and TLR-4 protein polymers were predicted and analyzed using the multitimer model in Alphafold v2.3.2, and docking visualization was performed in PyMOL v2.5.054. Using the PRODIGY web server, the binding strength, affinity, polymer structure, and dissociation constant were predicted based on the three-dimensional structure of the protein-protein complex. Finally, the molecular interactions within the protein were explored and visualized using the Arpeggio web server, and the interaction force between MEV and TLR-4 was evaluated. PRODIGY v2.0 predicted a strong binding affinity of -20.3 kcal / mol and a dissociation constant (M) of 1.3e-15 at 25.0°C. A dissociation constant less than 1.0e-8 indicates strong binding, less than 1.0e-4 indicates moderate binding, and greater than 1.0e-4 indicates weak binding, indicating that the binding force between MEV and TLR-4 is strong, and the two are tightly bound and have strong affinity. Visualization of protein interactions using PRODIGY v2.0 ( Figure 2 ), revealing the intermolecular interactions, including hydrogen bonds, van der Waals forces, charge interactions, aromatic ring atom interactions, and hydrophobic interactions. The analysis results showed that the strong intermolecular interactions between MEV and TLR-4 involved multiple mechanisms, including hydrogen bonds, van der Waals forces, charge interactions, aromatic ring atom interactions, and hydrophobic interactions.

[0088] Furthermore, the Gromacs software was used to simulate and study the interaction between tMEV and TLR-4 receptor. The corresponding graph was generated by calculating the RMSD value between MEV and TLR-4 ( Figure 3 -a). The analysis showed that the RMSD value between the protein and the small molecule was high within the first 20 ns of the simulation and then gradually increased. However, overall, the RMSD value was always within a narrow range, indicating that there was a stable binding interaction between the protein and the small molecule. Subsequently, the Gyrate value between the protein and the small molecule was calculated and visualized in the Gyrate plot ( Figure 3 -b). The results showed that the Gyrate value of MEV binding to TLR-4 decreased compared with that of a single protein molecule. This means that MEV binding resulted in a more compact TLR-4 structure and had minimal effect on the stability of the protein molecule. In addition, by comparing the MEV group and the TLR-4 group, SASA, which reflects the surface area of ​​the protein, was detected and the corresponding SASA graph was generated ( Figure 3 -c). The results showed that before MEV binds to TLR-4, the SASA of the protein increases and decreases after binding, indicating that the surface area of ​​the protein decreases after the small molecule binds. In addition, the HBonds between MEV and TLR-4 was simulated and evaluated, and the HBonds diagram was drawn ( Figure 3-d). The analysis showed that many hydrogen bonds were formed between MEV and TLR-4, mainly involving key protein residues and essential small molecule groups. Finally, the MMGBSA value of the protein-protein interaction remained stable throughout the simulation, maintaining at about -45 kJ / mol.

[0089] The above results all indicate that there are strong and stable protein-protein interactions between MEV and TLR-4. These interactions positively affect the overall structural stability, internal structural stability, compactness, surface properties and interaction strength.

[0090] Example 2 Specificity analysis of recombinant epitope antigen tMEV

[0091] Western blot analysis was performed on serum samples from patients with Mycobacterium tuberculosis and normal subjects to verify the specificity of the recombinant epitope antigen of the present invention. tMEV was separated on a 12% SDS-PAGE gel and then transferred to a PVDF membrane at a current of 280 mA. Western blot used a protein-free rapid blocking solution to block the membrane at 37°C for 15 minutes. The primary antibody was then incubated at 4°C overnight, and the secondary antibody was incubated for 1 hour before color development. The primary antibody used serum from patients with Mycobacterium tuberculosis positive patients and normal subjects, and the secondary antibody was a goat anti-human IgG antibody.

[0092] WB test results Figure 4 As shown in the figure, it can be seen that the serum of tuberculosis patients has an obvious binding band at 51kd, while the normal serum has no binding band, indicating that MEV can be recognized by the serum of pulmonary tuberculosis patients, but not by the serum of normal patients, indicating that it can bind to tuberculosis antibodies, has a certain specificity, and is an effective vaccine candidate protein.

[0093] Example 3 Analysis of the immune effect of the recombinant epitope antigen tMEV

[0094] Twenty-four 6-week-old BALB / c female mice were purchased from the Animal Experiment Center of Xinjiang Medical University and raised under standard conditions. The mice were randomly divided into 4 groups: control group (HC group), tMEV group, rBCG-tMEV group (subcutaneous injection of rBCG-tMEV) and BCG group (subcutaneous injection of BCG, REBIO, RC3452). The mice in the control group were subcutaneously injected with PBS (6 mice), tMEV group (6 mice), rBCG-tMEV 50μg (6 mice), and BCG group were subcutaneously injected with BCG 50μg (6 mice). Immunizations were performed at 0, 2, and 4 weeks, respectively, and the challenge experiment was performed 4 weeks after vaccination. The challenge strain was H37Rv. Four weeks after the last vaccination, 6 mice in each group were killed, and blood and spleen were taken for ELISA, ELISPOT and flow cytometry detection. Eight weeks after the challenge, 3 mice in each group were killed, and spleen and lung were taken to determine the bacterial load.

[0095] The rBCG-tMEV is BCG capable of secreting MEV prepared according to the method of Example 1.

[0096] 3.1 ELISA analysis

[0097] The serum IgG levels of mice in each group were measured by ELISA, confirming the generation of humoral immunity. The experiment was repeated 3 times, the OD405 value was obtained, and the average value was calculated to obtain the average IgG1 and IgG2a levels. At the same time, in order to evaluate the potential of the vaccine to induce specific humoral immune responses in immunized mice, serum IgG1 and IgG2a antibody titers were measured by ELISA. Optical density (OD) values ​​were recorded at 450nm, and values ​​exceeding 0.2 were considered to be effective antibody titers. Subsequently, the average logarithm (antibody titer) was calculated to express the results. The results showed that compared with the PBS group, the tMEV, BCG and rBCG-tMEV groups significantly induced the production of IgG1 ( Figure 5 B) and IgG2a ( Figure 5 C) Antibodies (P < 0.001), indicating that these vaccines can activate systemic humoral immunity. Notably, rBCG-tMEV induced no significant differences in IgG1 and IgG2a antibody levels compared with BCG; however, rBCG-tMEV did show higher production of IgG1 antibodies compared with BCG.

[0098] In mice, Th1 cells are responsible for the production of IgG2a antibodies, while Th2 cells are responsible for the production of IgG1 antibodies. Therefore, compared with the PBS group, tMEV induced a Th2-biased immune response, BCG induced a Th1-biased immune response, and rBCG-tMEV induced a mixed Th1 and Th2 immune response. In addition, the stimulation of IgG2a antibodies was positively correlated with the effectiveness of the vaccine. Both tMEV and rBCG-tMEV significantly induced higher levels of IgG2a antibodies than the PBS group (P<0.001), indicating that they may provide immune protection in mice ( Figure 5 A).

[0099] The above results show that the recombinant epitope antigen in the present invention is significantly higher than the level of the prior art in terms of both cellular immunity and humoral immunity, and is an ideal BCG vaccine antigen.

[0100] 3.2 Analysis of short-term cellular immunity

[0101] After confirming the expression of tMEV, we aimed to evaluate the immunogenicity of rBCG-tMEV as a vaccine candidate. To this end, we immunized mice with PBS, tMEV, BCG and rBCG-tMEV. It is generally accepted that the generation of long-lasting memory T cells against pathogens is crucial for the effectiveness of any successful vaccine. In order to facilitate the comparative analysis of the short-term cellular immunity levels of immunized mice, we isolated the spleens of mice under sterile conditions 4 weeks after the final immunization, prepared single-cell suspensions, and filtered them through a 70 μM mesh. The single-cell suspensions were labeled with anti-mouse fluorescent dye antibodies (Abs): CD3-APC, CD4-FITC, CD8-PE-Cy5.5, from BD Biosciences. Then, the cell number 1×10 6 Afterwards, the detection was performed on the Quantobio flow cytometer. Finally, FlowjoV 10 performed data processing.

[0102] The ratio of CD4+T cells to CD8+T cells in spleen cells was further evaluated ( Figure 6 AB). The results showed that tMEV, BCG, and rBCG-tMEV groups were able to activate cellular immunity compared with the PBS group. Among them, the CD4+T cell counts in the tMEV group, BCG group, and rBCG-tMEV group were significantly higher than those in the PBS group (P<0.001). Similarly, both BCG and rBCG-tMEV led to a significant increase in CD8+T cells compared with the PBS group (P<0.001). It is worth noting that although tMEV was also able to activate an increase in CD8+T cells, this increase was not statistically different from that observed in the PBS group. In addition, compared with the BCG group, the rBCG-tMEV group had a significant increase in CD4+T cells (P<0.001), although the number of CD4+T cells activated by tMEV was reduced. Similarly, compared with BCG, the rBCG-tMEV group had an increase in CD8+T cells, but the difference was not statistically significant. Therefore, the cellular immune response induced by rBCG-tMEV may exceed that induced by BCG, while the cellular immune response induced by tMEV may be inferior to that induced by BCG and rBCG-tMEV.

[0103] In general, tMEV has a stronger ability to activate CD4+T cells, and BCG has a stronger ability to activate CD8+T cells. rBCG-tMEV may integrate the advantages of both.

[0104] At the same time, we used the enzyme-linked immunosorbent spot (ELISPOT) assay to detect the level of specific antibody secreting cells. The spot test results are as follows: Figure 7As shown. The ELISPOT method is: add 100ul PBS, BCG (1×105CFU / ml), tMEV (5ug / ml), rBCG-tMEV (1×105CFU / ml) into the wells. At the same time, add 100ul (106cell / ml) spleen cell separation fluid into the experimental wells, add positive stimulation (ConA) into the positive control wells, and culture together for 24 hours. Add biotin antibody and antibody respectively, wash the cells and then detect. Each sample has 3 replicate wells. Finally, the ELISPOT plate counts the spots and records the spot parameters for statistical analysis.

[0105] The establishment of Th1 immune response is essential for fighting tuberculosis infection. Interferon gamma (IFN-γ) released by Th1 cells plays a key role in Mycobacterium tuberculosis (Mtb) infection by activating phagocytes. Four weeks after immunization, mouse spleen lymphocytes were collected and antigen-specifically stimulated with phosphate-buffered saline (PBS), tMEV, BCG, and rBCG-tMEV to study whether mouse spleen immunization can enhance IFN-γ secretion ( Figure 7 A). Compared with the PBS group, tMEV, BCG, and rBCG-tMEV all significantly stimulated spleen lymphocytes to produce more IFN-γ (P<0.001) ( Figure 7 A). Notably, rBCG-tMEV induced a significant increase in IFN-γ secretion by mouse splenic lymphocytes compared with BCG (P<0.001). Therefore, IFN-γ secreted by rBCG-tMEV activates macrophages and promotes the host response to Mtb infection.

[0106] Interleukin-2 (IL-2) is another Th1 cytokine that plays an important role in tuberculosis infection by promoting the growth of natural killer (NK) cells, enhancing the cytotoxicity and lethality of monocytes and macrophages, and participating in the eradication of Mycobacterium tuberculosis. Similarly, antigen-specific stimulation with PBS, tMEV, BCG, and rBCG-tMEV was performed to evaluate whether spleen immunization in mice could enhance IL-2 secretion ( Figure 5 B). Compared with the PBS group, tMEV, BCG, and rBCG-tMEV all significantly stimulated spleen lymphocytes to produce more IL-2 (P<0.001) ( Figure 7 B). In addition, compared with BCG, rBCG-tMEV induced a significant increase in IL-2 secretion in mouse splenic lymphocytes (P<0.001). Therefore, IL-2 secreted by rBCG-tMEV may promote the clearance of Mycobacterium tuberculosis by NK cells and monocytes and macrophages during Mycobacterium tuberculosis infection.

[0107] Overall, tMEV, BCG, and rBCG-tMEV showed the potential to enhance specific Th1 responses and activate cytotoxic T lymphocyte (CTL) responses, consistent with predictions from previous immune simulations.

[0108] 3.3 Analysis of spleen viable bacterial count

[0109] The presence of viable bacteria in the lungs and spleens of mice infected with Mycobacterium tuberculosis after immunization was evaluated 8 weeks later. The left lung (0.1 g) and part of the left spleen (0.1 g) of the mice were placed in a PANTA-containing chamber using Gentle MACS M-tubes (Miltenyi Biotec). TM The homogenate was diluted on 7H11 plates (BD Biosciences) and incubated at 37°C and 5% CO2 for about 14 days. CFU was calculated and the data were logarithmically transformed to display the data as log10 CFU. Figure 8 As shown. Compared with the PBS group, the colony forming units (CFU) in the lungs and spleens of the BCG group and rBCG-tMEV vaccine group were significantly reduced (P<0.05). The above results show that the rBCG-tMEV group has a good preventive effect on the mouse tuberculosis model.

[0110] It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation to the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein, on the contrary, the present invention can be extended to all other methods and applications with the same functions.

Claims

1. A BCG-based multi-epitope vaccine antigen, characterized in that The invention comprises a recombinant epitope polypeptide which is composed of a CD8+T cell epitope (CTL) polypeptide, a CD4+T cell epitope (HTL) polypeptide, a B cell epitope (LBEs) polypeptide, and a B cell conformational epitope (CBEs) polypeptide sequence connected in series, wherein: The CD8+T cell epitope (CTL) polypeptide is composed of the following amino acid sequences connected in series: SEQ ID No. 1SEAYQGVQQK; SEQ ID No. 2EAYQGVQQKW; SEQ ID No. 3ASTEGNVTGM; SEQ ID No. 4GTQVSDNALH; SEQ ID No. 5NASAQDQLHR; SEQ ID No. 6GANPHVVQAR; SEQ ID No. 7VQARPSVVPR; The CD4+T cell epitope (HTL) polypeptide is composed of the following amino acid sequences connected in series: SEQ ID No. 8QQWNFAGIEAAASA; SEQ ID No. 9TEQQWNFAGIEAAAS; SEQ ID No.10AGNAYAHAEAANAAR; SEQ ID No.11GNAYAHAEAANAARV; The B cell epitope (LBEs) polypeptide is composed of the following amino acid sequences connected in series: SEQ ID No.12GGSGSEAYQGVQQKWD; SEQ ID No.13EKMSHDPIAADIGTQV; SEQ ID No.14PEINSARIYAGAGSGP; SEQ ID No.15GAGMPAGMMGRGGANP; SEQ ID No.16PDSYPWVPSINPGLNF; SEQ ID No.17VPSINPGLNFYFGQPQ; The B cell conformational epitope (CBEs) polypeptide is composed of the following amino acid sequences connected in series: SEQ ID No. 18NIRQAGVQYS; SEQ ID No. 19AAWGGSGSEAYQ; SEQ ID No. 20VQYSRADEEQ; SEQ ID No. 21MAEMKTDAAT; SEQ ID No.22STAGSLQGQWRGAAGTAAQAAVVRFQE; SEQ ID No. 23AAWGGSGSEAYQGVQQ.

2. The multi-epitope vaccine antigen according to claim 1, characterized in that The recombinant epitope polypeptide sequence further comprises a connecting peptide sequence; Preferably, the connecting peptide is used to connect the polypeptide sequence in the CD8+T cell epitope (CTL) in series, and the amino acid sequence of the connecting peptide is shown in SEQ ID No. 24; Preferably, the connecting peptide is used to connect the polypeptide sequence in the CD4+T cell epitope (HTL) in series, and the amino acid sequence of the connecting peptide is shown in SEQ ID No. 25; Preferably, the connecting peptide is used to connect the polypeptide sequences in the B cell epitopes (LBEs) in series, and the amino acid sequence of the connecting peptide is shown in SEQ ID No. 26; Preferably, the connecting peptide is used to connect the polypeptide sequences in the B cell conformational epitopes (CBEs) in series, and optionally, the amino acid sequence of the connecting peptide is shown in SEQ ID No. 26; Preferably, the connecting peptide is used to connect the CD8+T cell epitope (CTL) polypeptide and the CD4+T cell epitope (HTL) polypeptide sequence, and the amino acid sequence of the connecting peptide is shown in SEQ ID No. 27; Preferably, the connecting peptide is used to connect the CD4+T cell epitope (HTL) polypeptide sequence and the B cell epitope (LBEs) polypeptide, the B cell epitope (LBEs) polypeptide and the B cell conformational epitope (CBEs) polypeptide sequence, and the amino acid sequence of the connecting peptide is shown as SEQ ID No.

26.

3. The multi-epitope vaccine antigen according to claim 2, characterized in that: The recombinant epitope polypeptide further comprises an immunomodulatory sequence, wherein the immunomodulatory sequence is a human β-defensin-3 adjuvant sequence; Preferably, the immunomodulatory sequence is the human β-defensin-3 adjuvant sequence as shown in SEQ ID No.

27. Preferably, the immunomodulatory sequence is connected to the N-terminus of the recombinant epitope polypeptide using a connecting peptide, and the connecting peptide sequence is shown in SEQ ID No.

28.

4. The multi-epitope vaccine antigen according to claim 3, characterized in that: The recombinant epitope polypeptide further comprises an adjuvant antibody response sequence, and the adjuvant antibody response sequence is a PADRE sequence; Preferably, the adjuvant antibody response sequence is shown as SEQ ID No. 29; Preferably, the adjuvant antibody response sequence is connected to the immunomodulatory sequence using a connecting peptide, and the connecting peptide is a sequence as shown in SEQ ID No.

30.

5. The multi-epitope vaccine antigen according to claim 4, characterized in that: The recombinant epitope polypeptide comprises a his-tag purification tag; preferably, the tag sequence is shown as SEQ ID No.

31.

6. The multi-epitope vaccine antigen according to claim 5, characterized in that: The recombinant epitope polypeptide sequence in the BCG multi-epitope vaccine is shown as SEQ ID No.

32.

7. The nucleotide sequence of the multi-epitope vaccine antigen according to claim 6 is shown in SEQ ID No.

33.

8. Use of the multi-epitope vaccine antigen according to any one of claims 1 to 7 in the preparation of a BCG preventive or therapeutic drug; preferably, the BCG preventive or therapeutic drug is a vaccine.

9. The multi-epitope vaccine according to claim 8, characterized in that Comprising the multi-epitope vaccine antigen according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier or agent.

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